Cytogenetic Methods

Cytogenetic methods František Šťáhlavský 1 Cytogenetic is a branch of genetics that is concerned with the study of the structure and function of the cell, especially the chromosomes. 1842 – first observations of chromosomes 1888 – used term chromosome (chroma=colour , soma=body ) 1902-04 – chromosomal inheritance theory 1950s – progress in methods (hypotonization,… 1956 – final determination of 2n in human 1968 – banding techniques 1990s – FISH techniques During this lecture, we will focus on Cytogenetic techniques. The cytogenetic is relatively old branch of science analyzing mainly the chromosomes and this research is closely related to the development of microscopes during the nineteenth century. The first microscoped chromosomes were observed like small dark dots inside the cells. Shortly after these findings the chromosomal inheritance theory was presented which means that the chromosomes hold genetic information and transfer this information equally to the new cells during the cell division. However, an early analysis used only histological sections that don’t provide a good resolution for chromosome observation. Therefore, it was not easy to count the diploid numbers and very often it was incorrect. This was improved during the fifties when new cytogenetic methods were developed. Since then it was possible to specify the number and morphology of chromosomes more precisely. For example, the correct number of chromosomes in human cell was specified in 1956. The second half of the last century became a cytogenetics boom. The new banding techniques were developed that enabled to identify homology of chromosomes, even between the species. The new era represents the implementation of Fluorescent in situ hybridization which is better known as FISH technigue. This technique enables to identify the location of specific genes. But even the standard cytogenetic techniques may also help you to identify changes of genome reorganization. Moreover, these techniques may be very fast and cheap. So, they are still very useful. 2 Functions of chromosomes (2x histons H2A, H2B, H3 a H4) - spatial distribution of genes - transport of genetic information during cell division - crossing-over during meiosis, EUCHROMATIN x HETEROCHROMATIN new genetic combinations genetic activity no genetic activity Drosophila melanogaster (2n = 8) On this slide, we need to repeat basic information about the chromosomes. Chromosomes are not only the DNA but they also contain different proteins (mainly several histones) that enable the spatial organization of the DNA and regulate also the activity of the genes. The chromosomes are usually condensed and visible as interphase nuclei in the cells. The interphase nucleus may seem to be a chaotic structure, but it is obviously not. The FISH techniques show that every chromosome has a specific position inside this interphase nucleus. Moreover, the chromosomes have not the same value regarding their genetic content. We distinguish Euchromatin with genetic activity and Heterochromatin with no genetic activity. Here are three main functions of chromosomes. The first is the specific localization of the genes. During the rearrangements of chromosomes, the genes may change their position and may be transferred to the new areas that may change their transcription activity. The second, chromosomes also guarantee equal separation of genetic information to new cells during cell division. And third during the meiosis, the crossing over between homologous chromosomes forms new genetic variability. 3 Melters et al. 2012 1 2 3X H holocentrics scorpion Tityus bahiensis (2n=5-19) Pseudoscorpion: Olpium turcicum: 2n = 7, X0 We distinguish two different types of chromosomes that differ in the organization of kinetochores. This structure is located on the surface of chromosomes and makes the connection to microtubules of spindle apparatus that guarantee equal separation of chromosomes to the new sister cells during cell division. In the monocentric chromosomes, the kinetochore is located only in a small area that we name centromere. This area is often visible as a constriction on the chromosome, so-called primary constriction. The second type is the holocentric chromosomes. In this case, the kinetochore is spread on the surface of the chromosome. This organization is beneficial when some fractures happen because than both fragments of chromosome include kinetochore for connection to microtubules and still, they may be equally separated during cell division. While in the monocentric chromosomes fragments without kinetochores are eliminated. It is still disputable which type of chromosomes is ancestral because both types are distributed equally all-over different organisms. 4 Monocentric chromosomes M SM ST T Holocentric (holokinetic) chromosomes www.metasystems-international.com/ikaros www.lucia.cz In monocentric chromosomes, we recognize four morphological types according to the position of the centromere. It is easy to qualify them as the ratio of long and short arms. The holocentric chromosomes have not any visible constriction. So, we don’t have a chance to specify the morphology of these chromosomes, we can measure only the length. It is a reason why it is more complicated to identify homologs in holocentrics without additional banding methods. You can use automatic software to sort and categorize the chromosomes, for example, IKAROS and LUCIA. However, these systems are developed mainly for human diagnostics and so they are not cheap. 5 http://imagej.nih.gov/ij/ http://rsb.info.nih.gov/ij/plugins/levan/levan.html http://www.drawid.xyz If you want to describe the karyotype you also can use free software. The main opportunity is plugin LEVAN for ImajeJ that sort the chromosomes according to your measurements of the arms of chromosomes. Similarly, you can use also DRAWID that enables also to create the ideogram. 6 Karyogram of scirpion Bothriurus rochensis Karyogram of Astyanax fasciatus deduced after conventional Idiogram (ideogram) Giemsa staining and double FISH using 5S (green) and 18S of scorpion Tityus trivittatus rDNA (red) probes. Here is not any mandatory style of visualization of karyotypes. You can find mainly two different types in the articles. The first type sorts the chromosome pairs according to the size and the morphology is specified usually separately in the text or table. The second type of visualization sorts the chromosome pairs firstly according to morphology than according to the size. You can find also ideograms (or idiograms) that represent schematic visualization of chromosomes. They may contain specific characteristics like the location of different bands of genes. 7 number of chromosomes from 2n=2 to 2n = 446 Plebicula atlantica Parascaris univalens, ants Myrmecia pilosula, M. croslandi, (males n=1) Márquez-Corro et al. 2018 The knowledge about the karyotypes is very useful, especially for species delimitation. It is known, that karyotypes of organisms differ sometimes very distinctly, even between closely related species. The variability of the basic characteristic such as the number of chromosomes is very wide within animals. The diploid numbers range from two chromosomes in some ants and mites to more than four hundred in one butterfly. It should be noted that ants have haplodiploidy, it means that males are haploid. So, the lowest number of chromosomes is in fact only one chromosome. The higher limit of chromosomes may be even higher in some polyploids. As I already told you, we recognize monocentric and holocentric chromosomes. The fragmentation is more probable in holocentric chromosomes. It is also the case of lycaenid butterfly Plebicula atlantica (it was demonstrated that this species is not polyploid but the chromosomes are fragmented). For many years it was presented that the holocentrics are more variable owing to the frequent fragmentation. Interestingly, the recent analysis shows that the diversification rates of karyotypes are similar between sister groups with monocentrics and holocentrics. 8 number of chromosomes Modal number of chromosomes Odonata n=13 Lepidoptera n=28-32 spiders birds n=39-42 Salticidae Diptera n=2-10 different families Iguanidae pseudoscorpions Despite the high variability of karyotypes, some groups may have a very stable number of chromosomes. And this most frequent number is called MODAL NUMBER. The number may be stable in some groups or may change during the evolution and then become constant again (as you can see in family Iguanidae). 9 Zima 2000 Fish app. 30000 species - 1700 karyotyped http://coleoguy.github.io/karyotypes/ Arachnids Karyotypes http://www.arthropodacytogenetics.bio.br/index.html For the utilization of the information about the karyotypes for example in the taxonomy, it is very important to know the variability of closely related taxa. It is relatively easy for example in mammals. At this moment we have information about the karyotypes in the majority of mammal species, including also the information about the intraspecific variability (especially in Europe). Unfortunately, we have not so much information in the remaining groups especially in invertebrates. Currently we have several online databases with the karyotypes of amphibians, beetles, polyneoptera, and some arachnids. 10 Euscorpius (Alpiscorpius) germanus group Scorpiones Now, I would like to show you some results of our team that use the cytogenetic characteristic in species delimitation of scorpions. The members of this arachnid order are very morphological uniform and this group contains

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